The present invention relates to a method for selecting solid tumours which are sensitive to anticancer treatment, which inhibits or prevents the HLA-G activity of said solid tumours, and to uses thereof.
Major histocompatibility complex (MHC) antigens are divided up into several classes, class I antigens (HLA-A, HLA-B and HLA-C) which exhibit 3 globular domains (xcex11, xcex12 and xcex13) and whose xcex13 domain is associated with xcex22 microglobulin, class II antigens (HLA-DP, HLA-DQ and HLA-DR) and class III antigens (complement).
Class I antigens comprise, besides the abovementioned antigens, other antigens, so-called unconventional class I antigens, and in particular the HLA-E, HLA-F, and HLA-G antigens; the latter, in particular, is expressed by extravillous trophoblasts of normal human placenta and thymic epithelial cells.
The sequence of the HLA-G gene (HLA-6.0 gene) was described by Geraghty et al. (Proc. Natl. Acad. Sci. USA, 1987, 84, 9145-9149): it comprises 4396 base pairs and exhibits an intron/exon organization which is homologous to that of the HLA-A, -B and -C genes. More specifically, this gene comprises 8 exons, 7 introns and a 3xe2x80x2 untranslated end; the 8 exons correspond respectively to: exon 1: signal sequence, exon 2: xcex11 extracellular domain, exon 3: xcex12, extracellular domain, exon 4: xcex13 extracellular domain, exon 5: transmembrane region, exon 6: cytoplasmic domain I, exon 7: cytoplasmic domain II (untranslated), exon 8: cytoplasmic domain III (untranslated) and 3xe2x80x2 untranslated region (Geraghty et al., mentioned above: Ellis; et al., J. Immunol., 1990, 144, 731-735; Kirszenbaum M. et al., Oncogeny of hematopoiesis. Aplastic anemia Eds. E. Gluckman, L. Coulombel, Colloque INSERM/John Libbey Eurotext Ltd). However, the HLA-G gene differs from the other class I genes in that the in-frame translation stop codon is located in the second codon of exon 6; consequently, the cytoplasmic region of the protein encoded by this HLA-6.0 gene is considerably shorter than the cytoplasmic regions of the HLA-A, -B and -C proteins.
These HLA-G antigens are essentially expressed by the cytotrophoblastic cells of the placenta and are considered to play a role in protecting the foetus (absence of rejection by the mother). In addition, since the HLA-G antigen is monomorphic, it may also be involved in placental cell growth or function (Kovats et al., Science, 1990 248, 220-223).
Other research relating to this unconventional class I antigen (Ishitani et al., Proc. Natl. Acad. all. Sci. USA, 1992, 89, 3947-3951) has shown that the primary transcript of the HLA-G gene can be spliced in w several ways, and produces at least 3 distinct mature mRNAs: the primary transcript of HLA-G provides a 1200-bp complete copy (G1), a 900-bp fragment (G2) and a 600-bp fragment (G3).
The G1 transcript does not comprise exon 7, and corresponds to the sequence described by Ellis et al. (mentioned above), i.e. it encodes a protein which comprises a leader sequence, three external domains, a transmembrane region and a cytoplasmic sequence. The G2 mRNA does not comprise exon 3, i.e. it encodes a protein in which the xcex11 and xcex13 domains are directly joined; the G3 mRNA contains neither exon 3 nor exon 4, i.e. it encodes a protein in which the xcex11 domain and the transmembrane sequence are directly joined.
The splicing which prevails so as to obtain the HLA-G2 antigen leads to the joining of an adenine (A) (originating from the domain encoding xcex11) with an AC sequence (derived from the domain encoding xcex13), which leads to the creation of an AAC (asparagine) codon in place of the GAC (aspartic acid) codon encountered at the start of the sequence encoding the xcex13 domain in HLA-G1.
The splicing generated so as to obtain HLA-G3 does not lead to the formation of a new codon in the splicing zone.
The authors of this article also analysed the various proteins expressed: the 3 mRNAs are translated into protein in the 221-G cell line.
Some of the inventors have shown the existence of other spliced forms of HLA-G mRNA: the HLA-G4 transcript which does not include exon 4; the HLA-G5 transcript which includes intron 4 between exons 4 and 5, thus causing a modification of the reading frame during the translation of this transcript and in particular the appearance of a stop codon after amino acid 21 of intron 4; and the HLA-G6 transcript which possesses intron 4, but has lost exon 3 (Kirszenbaum M. et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 4209-4213; European Application EP 0 677 582; Kirszenbaum M. et al., Human Immunol., 1995, 43, 237-241; Moreau P. et al., Human Immunol., 1995, 43, 231-236); they have also shown that these various transcripts are expressed in several types of foetal and adult human cells, in particular in lymphocytes (Kirszenbaum M. et al., Human Immunol., 1995, mentioned above; Moreau P. et al., Human Immunol. 1995, mentioned above).
Some of the inventors have also shown that NK cells express no HLA-G transcript (Teyssier M. et al., Nat. Immunol., 1995, 14, 262-270; Moreau P. et al., Human Immunol., 1997, 52, 41-46).
At least 6 different HLA-G mRNAs thus exist which potentially encode 6 protein isoforms of HLA-G, of which 4 are membrane-bound (HLA-G1, G2, G3 and G4) and 2 are soluble (G5 and G6).
Although the foetus can be considered to be a semi-allograft, the foetal cells survive and are not rejected by the mother; it has become apparent that the HLA-G molecules expressed at the surface of the trophoblasts protect the foetal cells against lysis by maternal natural killer (NK) cells from the uterine decidua and from peripheral blood (Carosella E. D. et al., C. R. Acad. Sci., 318, 827-830; Carosella E. D. et al., Immunol. Today, 1996, 407-409; Rouas-Freiss N. et al., PNAS, 1997, 94, 5249-5254).
Previous studies have shown that the expression of HLA-G molecules at the surface of transfected target cells makes it possible to protect said target cells against the lytic activity of NK cells from the decidual layer of the maternal endometrium (Chumbley G. et al., Cell Immunol., 1994, 155, 312-322; Deniz G. et al., J. Immunol., 1994, 152, 4255-4261; Rouas-Freiss N. et al., Proc. Natl. Acad. Sci., 1997, 94, 5249-5254). It should be noted that these target cells are obtained by transfection with vectors comprising either HLA-G genomic DNA which potentially generates all the alternative transcripts, or with vectors containing the HLA-G1 and HLA-G2 cDNAs encoding the HLA-G1 and HLA-G2 protein isoforms (European Patent Application No. 0 677 582 and Application PCT/FR98/00333).
NK cells express receptors for class I MHC molecules (killer inhibitory receptors or KIR, or NKIR for NK inhibitory receptors) which are responsible for the inhibition of cytotoxicity when these HLA molecules, acting as ligands, are recognized by these receptors; for example, N. Rouas-Freiss et al., (Proc. Natl. Acad. Sci., 1997, 94, 5249-5254) showed that the expression of HLA-G protected K562 (human erythroleukaemia cell line) target cells transfected with the HLA-G1 and G2 isoforms against lysis. These cells are usually sensitive to NK cells.
These results testify to the fundamental role of the HLA-G molecule as an immunotolerance antigen. These results have been broadened to all of the membrane-bound isoforms. The cDNAs encoding the HLA-G1, G2, G3 and G4 isoforms which are expressed, after transfection, in various cell types, in particular transfected K562 cells and M8 tumour cells, inhibit NK and CTL cytotoxic functions.
Given the important role that the HLA-G molecule may play, the inventors, continuing with their work, more particularly studied tumour cells, and gave themselves in particular the aim of providing tools for selecting solid tumours which are sensitive to a treatment which inhibits the HLA-G antigens present in particular on certain tumours.
The subject of the present invention is a method for establishing the HLA-G transcription profile of a solid tumour with a view to selecting a treatment which is suited to said tumour and/or with a view to monitoring the evolution of said tumour, characterized in that it comprises:
(i) the removal of a tumour sample;
(ii) the extraction of the mRNA from said sample; a modified Chomczynski and Sacchi method using the RNA reagent NOW (Ozyme, France) can in particular be used;
(iii) the reverse transcription (RT) of said RNA;
(iv) the successive or simultaneous amplifications of the cDNAs obtained in (iii), in the presence of primers specific for each HLA-G isoform, and the analysis of the amplification products obtained by electrophoresis and/or specific hybridization and
(v) the establishment of the HLA-G transcription profile of said sample.
Preferably, the reverse transcriptions are primed with oligo-dTs on mRNA which is denatured in advance, for example at 65xc2x0 C., in the presence of a reverse transcriptase such as M-MLV reverse transcriptase (Gibco-BRL, Life technologies).
Also preferably, the cDNA amplification is carried out by polymerase chain reaction (PCR) using primers specific for the various HLA-G isoforms, in accordance with the following tables: